This invention relates to a retro-inverso analogue of thymosin alpha1 and its hybrids, native/retro-inverso thymosin alpha 1 hybrids, which are immunomodulators with high resistance to enzymatic hydrolysis and have prolonged in vivo activity, and pharmaceutical compositions containing them and methods for using them.
A standard cell-free protein extract preparation from the thymus gland, known as thymosin fraction V (TF5) (U.S. Pat. No. 4,082,737), was demonstrated to be a potent immunopotentiating preparation. TF 5 can suppress to various extents immune deficiency diseases and can also act in lieu of the thymus gland to reconstitute immune functions in thymic deprived and/or immunodeprived individuals (Wara et al., N. Engl. J. Med 292: 70, 1975). Analytical polyacrylamide gel electrophoresis and isoelectric focusing have demonstrated that TF5 consists of a number of polypeptides termed thymosin, with molecular weights ranging from 1,000 to 15,000.
The first of these peptides to be purified to homogeneity and sequenced from TF5 was called thymosin alpha 1 (TM-alpha1) (Goldstein et al., Proc. Natl. Acad. Sci. 74:725, 1977; U.S. Pat. No. 4,079,127). The chemical synthesis of TM-alpha1 by solution and solid phase synthesis techniques is described in U.S. Pat. Nos. 4,148,788 and 5,856,440. Identical to the native TM-alpha1 in the biological potent and amino acid sequence with the lack of the N-terminal acetyl group, recombinant TM-alpha1 can be produced in E. coli by recombinant DNA cloning techniques (Wetzel et al., Biochemistry 19:6096, 1980). TM-alpha1 analogs and derivatives also have been produced, U.S. Pat. Nos. 4,116,951 and 5,512,656. TM-alpha1 is a 28 amino acid acidic peptide with a molecular weight of 3,100 and a pI in the range of 4.0-4.3. TM-alpha1 maintains many of the biologic effects of TF5 and has been found to be 10 to 1,000 times more active than TF5 in a number of bioassay systems designed to measure the maturation and function of T lymphocytes.
TM-alpha1 potentiates the immune system by stimulating alpha- and gamma-interferon production, increasing production of T-cell growth factor and macrophage migration inhibitory factor, increasing T-cell numbers, inducing lymphocyte maturation and differentiation and expression of interleukin-2 receptors, and improving T-cell helper cell activity (Marshall et al., J. Immunol. 126:741, 1981; Mutchnick et al., Clin. Immunol. Immunopathol. 23:626, 1982; Low et al., Thymus 6:27,1984; Sztein et al., Proc. Natl. Acad. Sci. U.S.A. 83:6107, 1986; Serrate et al., J. Immunol. 1939:2338,1987; Baxevanis et al., Immunopharm. 13:133, 1987). TM-alpha1 is currently undergoing clinical trials in the U.S.A. as an immunomodulator in cancer patients, in individuals with chronic active hepatitis B, and as an immunoenhancer of vaccines in immunocompromised individuals. (Goldstein, A. L., Cancer Invest. 12:545, 1994; Lopez et al., Ann. Oncol. 5:741, 1994; Garaci et al., Eur. J. Cancer. 31A:2403,1995; Garaci et al., Mech. Ageing. Dev. 96:103, 1997; Bonkovsky, H. L., Hepatology 26(3 Suppl 1):143S, 1997; Liaw, Y. F., J. Gastroenterol. Hepatol. 12:S346, 1997). TM-alpha1 has been approved for use in the treatment of hepatitis B in many Asian countries.
However, like most endogenous and exogenous biologically active peptides, which are highly susceptible to proteolysis by naturally occurring aminopeptidases, TM-alpha1 also has a short half-life in vivo (Rost, K., Int. J. Clin. Pharmacol. Ther. 37:51, 1999).
Therefore, there remains a need to find new derivatives of TM-alpha1 that exhibit greater stability and activity.